Cryopreserved sperm from non-human primates have been used for AI and IVF, but macaque sperm, like human sperm, have highly variable cryoprotection requirements depending on the individual sperm donor. This biological variability has restricted progress in developing a cryopreservation protocol that preserves sperm motility in all semen samples. This significant problem is likely to be exacerbated by significant variability in the cryoprotection requirements of sperm from genetically altered or unique individuals. The long-range goal of this proposal is to assure the preservation of valuable primate models of human disease. The principal objective is to develop a clear understanding of the fundamental cryobiology of macaque sperm. The second objective is to develop a rational and reliable method for cryopreservation of macaque sperm. The specific goals of this project are to determine the major aspects of biochemical composition and biophysical characteristics of macaque sperm, the extent of osmotic stress-induced cell damage in macaque sperm, the effects from temperature on spermatozoal responses to osmotic stress, and to determine whether cryoprotectant exposure can modulate adverse cellular properties resulting from cooling and osmotic stress. Rational development of an optimized cryopreservation protocol will then be developed based on the fundamental knowledge of macaque sperm generated from the proposed experiments. This project has a highly qualified research team comprised of cell biologists and cryobiologists from the University of California, Davis and the University of Minnesota. The project will utilize numerous techniques to accomplish these goals. Fourier-transform infrared spectroscopy will be used to assess sperm membrane phase transitions. Computerized sperm motility analysis, epifluorescence and confocal microscopy, flow cytometry, differential scanning calorimetry, and cell volumetric analysis will be used to evaluate sperm functional parameters and sublethal damage to macaque sperm. Mathematical modeling will then be used to construct optimized cryopreservation protocols.